Detection of alcaline isophosphatases by electrophoresis

ABSTRACT

The invention concerns a process for separating alkaline phosphatase (ALP) isoenzymes from a biological sample by electrophoresis, characterized in that the electrophoresis is carried out on an electrophoresis support after depositing a solution of lectin onto the electrophoresis support in a predetermined localized zone, under conditions which permit interaction between said lectin and the ALP isoenzymes contained in the analyzed biological sample, deposition of the lectin solution further being carried out under conditions which are suitable to allow separation of the ALP isoenzymes constituted by the osseous fraction and by the hepatic fraction.

BACKGROUND OF THE INVENTION

Alkaline phosphatase (EC 3.1.3.1.) (abbreviation:ALP) is a metalloenzymeconsisting of a group of isoenzymes present in different tissues ofanimal organisms and in particular in man.

Alkaline phosphatase isoenzymes are important in protocols fordiagnosing different conditions in adults or in children, and a numberof methods have been proposed for separating and assaying suchisoenzymes. ALP isoenzymes can be divided into four classes:non-specific tissue (bone, liver and kidney), adult intestinal, foetalintestinal, and placental. A number of variations can exist within asingle class, namely:

hepatic: hepatic 1 (H1), hepatic 2 (H2);

ultrafast (UF);

osseous (Os);

placental: placental 1 (P1), placental 2 (P2);

intestinal: intestinal 1 (I1), intestinal 2 (I2), intestinal 3 (I3).

Thus nine principal fractions can be distinguished which have to beseparated, identified and quantified in particular for their detectioninto hepatic and biliary disorders and into certain bone diseases,including osseous tumours or Paget's disease.

Reference will occasionally be made below to the term “fraction” todesignate a class of ALP isoenzymes or a particular variant within aclass of isoenzymes. On the electrophoresis support, a “fraction”corresponds to a band revealed after migration.

The most frequent routine analysis carried out on alkaline phosphataseisoenzymes consists of measuring the total enzymatic activity using asubstrate of this enzyme, generally para-nitrophenylphosphate. Thatmethod, however, cannot determine the levels of the differentisoenzymes.

The principal method for separative analysis of such compounds useselectrophoretic techniques. Isoelectrofocussing is occasionally used andcan separate 10 to 20 bands depending on the procedure used. Identifyingall of the bands is difficult, rendering clinical interpretationextremely awkward.

Zone electrophoresis enables a good separation of the principal forms ofthe isophosphatases. However, certain fractions are superimposed, inparticular the Os, H1 and P1 fractions, and thus complementarytreatments have to be carried out to separate and identify them. Suchtreatments must be carried out on the biological test samples to betested before depositing them onto the gel.

Such treatments consist, for example, of thermal denaturing, incubationwith specific inhibitors such as urea, amino acids, etc., enzymaticincubation with neuraminidase, ficin, phospholipase C, incubation withspecific antiplacental or anti-intestinal antisera.

Several separation procedures which are in current use have been dealtwith by Van Hoof V. O., De Broe Marc, E., Clinical Laboratory Sciences,vol. 31, issue 3 1994, “Interpretation and clinical significance ofalkaline phosphatase isoenzyme patterns”.

One particular procedure has been proposed in U.S. Pat. No. 5,264,098which describes the separation of ALP isoenzymes using a gelelectrophoresis reaction employing a gel buffer containing at least onenon ionic detergent and an anionic detergent.

Available treatments for identifying and quantifying ALP isoenzymes havecertain disadvantages as regards routine analysis. In addition to highcosts, they can on the one hand be long and can considerably complicatemanipulation, and on the other hand, complete determination (of all ofthe isoenzymes) necessitates a plurality of treatments (2 or 3) for asingle sample, limiting the number of samples which can besimultaneously analysed on the one gel.

Other treatments have been proposed which, for example, recommendtreating the sample prior to loading onto the electrophoresis gel. Inthis regards, the action of the WGA lectin (wheat germ agglutinin) isparticularly interesting (see Sidney B. Rosalki, A. Ying Foo, ClinicalChemistry, 30/7, p. 1182-1186, 1984, “Two methods for separating andquantifying bone and liver alkaline phosphatase isoenzyme in plasma”,European patent EP-A-0 131 606 dated May 11, 1986). EP-A-0 131 606describes the differential detection of bone and liver ALP isoenzymecomprising treating the test sample with lectin, then incubating themixture obtained followed by separating the ALP bound to the lectin fromthe fraction containing free ALP and determining the ALP activity in oneof the two media or in both. In a particular implementation of thatpatent, the two fractions (ALP bound to lectin and free ALP) areseparated by electrophoresis.

With the exception of the intestinal forms, all isophosphatases possesssialic acids and are thus affected by a treatment with WGA lectin to agreater or lesser extent. The osseous fraction is the most sialated andthus is affected the most by this treatment, which under suitableconditions retards its mobility and thus causes it to precipitate in azone which is distinct from the zone where the hepatic fraction islocated.

In order to render ALP isoenzyme precipitation more selective towardsthe osseous isoenzyme, certain authors have used detergents such asTriton X100 (Rosalki). However, despite the presence of such detergents,residual interactions of the WGA lectin with other isophosphatasessubsist, which cause co-precipitation of such fractions with the osseousfraction.

In addition to this lack of specificity, a further disadvantage of thistechnique is to render the analysis considerably more complicated.

The publication by Rosalki S. B. et al, cited above, alternativelyproposes incorporating lectin into the buffer used to impregnate theelectrophoresis gel prior to using this gel. This dispenses with priortreatment of the sample. In that case, the majority of the osseousfraction is precipitated close to where the sample has been loaded. Themobility of all of the other isoenzymes with the exception of intestinalisoenzymes is affected by the action of the WGA lectin despite thepresence of the detergents mentioned above.

In the context of that treatment, the properties of the lectin used areits ability to interact specifically with the ALP isoenzymes whichcontain sialic acids.

SUMMARY OF THE INVENTION

The present invention proposes means for at least partially overcomingthe disadvantages stated in prior art methods. In particular, theinvention defines a method enabling separation and identification of ALPisoenzymes which is improved as regards specificity and sensitivity.

The present invention also provides consumers, in particular clinicians,with a process for separating, identifying and quantifying the principalalkaline isophosphatases, which process can be carried out in a singlestep on a single electrophoresis support which is easy to produce.

The invention thus proposes a novel process for separating andidentifying ALP isoenzymes by electrophoresis, characterized in that thelectin is deposited on the electrophoresis support in a localisedmanner.

The lectin deposited in solution in a localised manner can diffuse intothe support while remaining localised in a determined zone of thissupport during electrophoretic migration.

The deposit in question, located close to the zone where the sample isdeposited, is distinguished from the uniform loading over an extendedzone or over the whole of the electrophoresis support as described inthe prior art.

The invention thus provides a process for separating alkalinephosphatase isoenzymes from a biological sample by electrophoresis,characterized in that the electrophoresis reaction is carried out on anelectrophoresis support after depositing a solution of lectin onto theelectrophoresis support in a given zone, under conditions which permitinteraction between said lectin and the ALP isoenzymes contained in theanalysed biological sample, deposition of the lectin solution furtherbeing carried out under conditions which are suitable to allowseparation of the ALP isoenzymes constituted by the osseous fraction andby the hepatic fraction.

The interaction in question leads to the formation of a complex betweenthe lectin and the ALP isoenzyme until equilibrium is obtained.

The process of the invention enables the osseous fraction of the ALP tobe acted on in a manner which is more specific than on the other ALPfractions because of the reaction of this fraction with the lectin.

The biological sample analysed can be any biological sample which maycontain ALP, in particular a biological fluid sample such as a serum orplasma sample, or possibly a tissue sample removed from a patient.

In the invention, electrophoresis is carried out on any suitableelectrophoresis support, in particular on a gel, more particularly on anagarose or a polyacrylamide gel, or on a porous membrane, in particularmade of cellulose acetate.

The particular conditions defined above for carrying out theelectrophoresis of the invention can be applied in the context of knownelectrophoresis methods which may or may not be automated.

The localised lectin deposit zone is determined as a function of thedirection of migration of the sample and the lectin. Thus the lectindeposit zone is selected such that, during migration, the sampletraverses the lectin, the mobility of the latter during migration havingbeen taken into consideration. Similarly, the final position normallyreached by the other ALP isoenzymes is taken into consideration in orderto determine the lectin deposit zone with respect to that of the sample.In practice, the lectin and the sample are 1 to 10 mm apart,advantageously 5 mm, when the sample is loaded.

The other conditions for localised depositing of the lectin, such as theconcentration of the lectin, the time for application to theelectrophoresis support, are determined such that they enable osseousand hepatic ALP isoenzymes to be separated during electrophoresis underthe migration reaction conditions.

In other words, once the parameters of electrophoresis have beendetermined particularly as regards depositing the lectin, the process ofthe invention can separate the osseous and hepatic isoenzymes underconditions which are satisfactory for identifying them with respect tothe other ALP isoenzymes, and preferably to quantify them. The mobilityof the other ALP isoenzymes, affected during passage through the zonewhere the lectin is present, returns to normal outside this zone.

Since the osseous ALP fraction is more sialated, its electrophoreticmigration is the most affected by the passage of the sample through thelectin deposited on the support. As a result it is precipitated in azone which may be distinguished from the migration zone of other ALPisoenzymes.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a preferred embodiment of the invention, a process for separatingalkaline phosphatase (ALP) isoenzymes by electrophoresis ischaracterized in that it comprises:

depositing the biological sample containing the ALP isoenzymes to beseparated on the electrophoresis support;

depositing a lectin solution which can interact with the ALP isoenzymescontained in the sample on the electrophoresis support;

applying an electric field to permit electrophoretic separation bymigration of the ALP isoenzymes under conditions which can permitdifferential separation of the osseous and hepatic ALP fractions;

revealing the separated ALP isoenzymes.

Revealing is carried out using any known means, preferably using an ALPsubstrate.

At the end of the steps resulting in separation of the ALP isoenzymes onthe electrophoresis support, a quantitative analysis of the differentseparated isoenzymes, or certain of them, can be carried out.

Different methods can be used to quantify the ALP isoenzymes detected atthe end of electrophoresis. As an example, the densitometry of theelectrophoresis support can be measured after staining the separated ALPfractions using an ALP substrate.

When the osseous ALP fraction is in excess with respect to the fixingcapacity of the lectin deposited on the gel, the lectin may notprecipitate the whole of this fraction. In this case, the nonprecipitated portion continues to migrate with the other fractions, andcan be found in the form of a smear close to the subjacent isoenzymes,in particular H2, I1, I2 and I3. In this case, the different isoenzymescan be quantified by depositing the same sample a second time on thesame support, in the absence of lectin. On the “no lectin” pattern, thepercentages of the H1 Os P1 block and the separated fractions H2, I1,P2, I2, I3 and UF are determined. The percentage of the Os fraction isobtained by deducting the percentages of H1 and P1 determined from the“with lectin” profile from the H1 Os P1 block.

Within the context of the definitions given above, when carrying outelectrophoresis in the presence of a localised deposit of lectin on theelectrophoresis support, the lectin and the sample can be depositedsimultaneously on the electrophoresis support.

In another embodiment of the invention, the sample and the lectin aredeposited at different times.

Further, the sample and the lectin solution can respectively bedeposited on the electrophoresis support over periods which may beidentical or different.

Advantageously, within the context of the definitions given above, ifnecessary taken in combination, the lectin and the sample are depositedover a substantially identical period of time, and preferablysimultaneously for practical and economic reasons.

For a given sample and a determined concentration of lectin in thesolution, the sample application and lectin application period, and thusthe quantity of sample and lectin deposited on the electrophoresissupport, is selected so as to precipitate the Os fraction of the ALPisoenzymes such that the osseous and hepatic fractions are separatedduring migration. To determine the concentration of the lectin solutionand the period over which the solution is applied to the electrophoresissupport, the temperature reached by the support during migration is alsotaken into account.

Indeed, since the interaction of the lectin with the osseous ALPfraction is dependent on temperature, the temperature of theelectrophoresis support during the migration step must be taken intoaccount. A reduction in the lectin-osseous ALP interaction due to anincrease in the temperature of the electrophoresis support can becompensated for by an increase in the quantity of lectin deposited, forexample by increasing the concentration of the lectin solution used.

These parameters can be determined in the light of the indications belowand the values given in the examples and can if necessary be adapted tothe selected electrophoresis conditions, by carrying out tests such asthose which are given in the examples below.

The sample application and/or lectin solution application period canvary and can in particular be in the range of 5 to 20 min, preferably 15minutes, for the sample and/or for the lectin.

Depositing can be carried out using any known manual or automated means,for example using “comb” type applicators, for example the devicesdescribed in European patent application EP-A-0 493 996.

The lectin used is in the form of an aqueous solution.

Thus the concentration of lectin deposited on the electrophoresissupport used under normal conditions is in the range of 0.1 mg/ml to thelimit of solubility of lectin in water. This concentration isadvantageously in the range of 0.5 to 15 mg/ml, preferably in the rangeof 1 to 10 mg/ml.

In a particular implementation of the invention, the process ischaracterized in that when the lectin concentration is in the range of 1to 10 mg/ml, the migration temperature is respectively in the range of18° C. to 53° C. These conditions are applicable inter alia todepositing a lectin solution over a period of close to about 15 minuteswith a comb type applicator as described in European patent applicationEP-A-0 493 996.

The invention particularly concerns an implementation of anelectrophoresis process in which, when the ALP isoenzymes present in thetest sample have an electrophoretic mobility in the direction of theanode, the lectin is deposited between the anode and the sample depositzone.

Advantageously in this case, the lectin is deposited between thebiological sample deposit point and the zone normally occupied byintestinal fraction 3 (I3) at the end of the migration step, when thisfraction is present in the sample.

Different lectins can be used in the invention. Lectins are proteinswhich fix sialated groups. Lectins which can be used in the presentinvention which can be cited include wheat germ (Triticum vulgaris)lectin, or WGA (Wheat Germ Agglutinin). WGA lectin can be obtained fromSigma, Pharmacia, etc.

The invention also concerns a process satisfying the definitions givenabove, taken separately or in combination, in which separation offractions other than the osseous and hepatic 1 of ALP is improved.

The invention thus makes available, under conditions which arecompatible with routine laboratory analyses, means for in vitrodetection in a biological sample of the abnormal presence of one or moreALP isoenzymes.

This process can in particular be carried out during a protocol fordiagnosing a hepatic or biliary disorder corresponding to the abnormalpresence of the hepatic ALP fraction. The invention can also enable theabnormal presence of the osseous fraction to be researched in thecontext of the diagnosis of osseous disorders.

The invention also concerns kits for carrying out separation offractions constituted by ALP isoenzymes by electrophoresis.

The process of the invention thus has the advantage of using a singleelectrophoresis support, without prior treatment of the sample, to carryout qualitative determination of all of the ALP isoenzymes in onedeposit and their quantitative determination in two deposits onto asingle support. The electrophoresis support is free of lectin prior toits use, which enables it to be stored under the usual temperatureconditions and does not change its manufacturing cost.

A kit comprises, for example:

an electrophoresis support comprising a porous material suitable fordepositing a biological sample to be analysed and for carrying outelectrophoretic migration;

a solution of lectin in a concentration in the range of 0.1 mg/ml to 15mg/ml, advantageously in the range of 1 to 10 mg/ml, preferably in therange of 1 to 10 mg/ml.

The kit of the invention can also comprise the buffer or buffersrequired for the electrophoresis reaction. It can also contain ALPactivity revealing reagents.

Further advantages and features of the invention will become more clearfrom the following examples.

EXAMPLES

Principles of the Reaction Using Lectin When it is Uniformly Distributedin the Electrophoresis Gel

The interaction of the WGA lectin with a slightly sialatedisophosphatase can be represented by the equilibrium:

Isophosphatase+WGA⇄{isophosphatase-WGA} complex  (A)

Under normal electrophoresis conditions, namely at a basic pH,isophosphatases have an anodic mobility while lectin has a slightlycathodic mobility. The anodic mobility of the {isophosphatase-WGA}complex will thus be lower than that of free isophosphatase.

During the whole of the migration period, the isophosphatase willinteract with the WGA lectin in accordance with the equilibrium ofreaction (A) and thus will be slowed down.

Thus the pattern obtained on the gel with no WGA lectin where, under thegiven conditions, all fractions are separated with the exception of theblock constituted by the H1, Os (possibly P1) isoenzymes, will lead, fora gel incorporating WGA lectin over its entire surface, to a patternwhere the H1 (possibly P1) will be disengaged from the osseous but wherethe HI, P1 fractions on the one hand and the H2, I2 fractions on theother hand, which are resolved on the gel without WGA lectin, will bemerged. It would then be necessary, even for a simple qualitativeestimation of the isoenzymes of a sample, to carry out electrophoresison two types of gel, which would therefore complicate the analysis.

In addition to this disadvantage, WGA lectin is heat sensitive whichrenders the production of gels containing it very difficult; further,consumers are obliged to store these gels between 40° C. and 80° C. topreserve their performance intact.

Principles of the Reaction of the Invention, Using Lectin Deposited Ontothe Electrophoresis Gel in a Localised Manner

In the invention, a solution of WGA lectin is deposited in front of thesample, i.e., between the sample and the anode. The electrophoresis gelis thus not impregnated with lectin over its entire surface. The WGAlectin may or may not be deposited simultaneously with the sample. Onceboth deposits have been carried out, the voltage is applied to obtainelectrophoretic separation.

Under these conditions, the major portion of the Os fractionprecipitates out when it goes through the zone where the WGA lectin hasbeen deposited. The electrophoretic mobility of the other isophosphatasefractions is affected by the WGA lectin in accordance with theequilibrium of the reaction:

Isophosphatase+WGA⇄{isophosphatase-WGA} complex,

but only when traversing the zone containing the WGA lectin. This zoneis very reduced in size (<1 mm) which means that the profile remainspractically identical to that obtained in the absence of lectin, withthe exception of the osseous fraction which is disengaged from the H1and P1 fractions. Under these conditions, electrophoresis of the samplewith the deposited lectin in front of the sample permits qualitativeanalysis on a single gel in a single step: all of the isoenzymes can beidentified from their position and without the need for complementarytreatments to be carried out on the sample.

In the case when the different isoenzymes which have been separated andrevealed by a suitable reagent have to be quantified, account must betaken of the fact that precipitation of the osseous fraction by the WGAlectin is not complete, in particular in the case where this osseousfraction is highly increased. A portion of the osseous alkalinephosphatase molecules escape precipitation when going through the lectindeposit. The Os-WGA bond is, however, sufficient for thesenon-precipitated osseous alkaline phosphatase molecules to entrain WGAlectin with them. They are thus sufficiently slowed so that they do notreach the H1P1 zone. Thus in addition to the precipitation curve of theosseous fraction, a smear of osseous alkaline phosphatase is obtainedwhich reaches to the zone to which the H2 migrates. Under theseconditions, quantification of the isoenzymes subjacent to this streak,namely the isoenzymes H2, I1, I2, I3, is disturbed by this smear.

To enable quantification in a situation where the quantity of the Osfraction risks being higher than that which can precipitate, twodeposits of the same sample should be made side by side, one with thelectin deposit, the other without. The H1 and P1 fractions can bequantified on the pattern with the WGA lectin. The percentages of the HiOs P1 block and the separated H2, I1, P2, I2, I3 and UF fractions aredetermined using the pattern with no lectin. The percentage of the Osfraction is obtained by subtracting the H1 and P1 percentages determinedusing the profile with lectin from the H1 Os P1 block.

Thus a single gel can be used to determine, in a single step with twoloads, the percentage of all of the isoenzymes with no prior treatmentof the sample.

Concentration of Lectin and Temperature of Electrophoresis.

The concentration of lectin to be used and the migration temperature(temperature of the electrophoresis support) are closely linked. Theequilibrium for formation of the isophosphatase-WGA lectin complex isindeed temperature-dependent. Thus increasing the temperature encouragesdissociation of the complex. The lectin concentration must be increasedto obtain the same precipitating power of the osseous fraction.

In practice, the concentration of the lectin to be used is a function ofthe gel and can be selected using the following table:

Temperature of gel during migration Concentration of lectin, ° C. mg/ml18 1 28 2 38 3 48 5 53 10

Thus if the process is carried out in accordance with the invention withan electrophoresis system which cannot control the migration temperature(Example 1), the lectin concentration has to be increased to takeaccount of the maximum temperature achieved in the gel during migration.This temperature depends on a number of parameters such as the ionicstrength and the dimensions of the gel, the migration parameters and theexternal temperature during migration. Under the conditions used inExample 1, the maximum temperature reached by the gel is close to 38°C., and thus a lectin concentration of 3 mg/ml is used. In Example 2,the instrument can regulate the electrophoresis temperature at 20° C.However, when the electrophoresis is carried out at a constant power of20 W, the effective temperature of the gel is 28° C. As a result, theconcentration of lectin necessary to obtain the desired effect is 2mg/ml.

Position and Duration of Lectin Depositing

Under the pH conditions (basic) used for electrophoresis, the mobilityof all of the alkaline isophosphatases is in the anode direction; incontrast, lectin has a very slight mobility in the cathode direction. Inorder for the osseous fraction to encounter the lectin, the lectin hasto be deposited between the sample and the anode. More precisely, itmust be deposited at a distance which is less than or equal to thattraversed by the osseous fraction under the migration conditions usedand in the absence of lectin. However, in order to prevent precipitationof the osseous fraction in a zone already occupied by other fractions,it appears more judicious to deposit the lectin at a distance betweenthe sample and the position which the I3 fraction occupies at the end ofmigration. In practice, the lectin is deposited in front of the sampleat a distance in the range of 1 to 10 mm, preferably close to 5 mm.

The lectin is deposited at the same time or after the sample. The two donot have to be deposited simultaneously. However, if the sample and thelectin are not deposited at the same time, care must be taken to preventdiffusion of the first deposit when depositing the second. For thisreason it is easier to deposit the sample and the lectin simultaneouslyand over the same period. Further, the two deposits are parallel to eachother and perpendicular to the direction of migration.

Example n° 1

Gel with the following composition:

Agarose 1% Tris 0.03 M Sodium Barbital 0.025 M Barbital acid 0.005 MSodium azide 1 g/l Triton X 100 10 g/l Nonidet NP 40 5 g/l

40 ml of demineralised water and 0.5 g of agarose were introduced into a100 ml Erlenmeyer flask. After boiling for 5 minutes with constantstirring, the agarose had dissolved to produce a perfectly clearsolution. The temperature of this solution was reduced to 50° C. in athermostated bath. 10 ml of a concentrated buffer solution containing 18g/l of Tris, 27.75 g/l of sodium Barbital, 4.6 g/l of Barbital acid, 5g/l of sodium azide, 50 g/l of Triton X 100 and 25 g/l of Nonidet NP 40were introduced into a 50 ml Erlenmeyer flask. This solution wasmaintained at 50° C. in the thermostatted bath.

The pre-heated buffer was added to the agarose solution. It washomogenised and maintained at 50° C.

5 ml of the above solution, removed with a pipette, was then uniformlypoured onto a 10×8 cm hydrophilic plastic sheet.

After gelling and stabilisation, the gel could be used. Fresh sera to beanalysed were deposited in 2 adjacent deposits, 2.5 cm from the edge, onthe cathode side. A 3 mg/ml solution of Wheat Germ Agglutinin (WGA)lectin was applied simultaneously and over the same period 32 mm infront of one of the 2 deposits of each sample, i.e., between the sampleand the anode. The application time could be 15 minutes for each depositmade with a microporous membrane applicator as sold by Sebia, describedin European patent application EP-A-0 493 996.

The alkaline isophosphatases were separated by electrophoresis in avessel the tanks of which contained a Tris 0.003 M, sodium Barbital0.025 M, Barbital acid 0.005 M, sodium azide 0.1 g/l buffer for a periodof 50 minutes at a constant voltage of 100 V.

Incubating the gel with a conventional substrate for this enzyme (forexample bromochloroindolyl phosphate and nitrobluetetrazolium) thenrevealed the alkaline phosphatase activities. A blue stain was thusobtained at the location of each isophosphatase fraction, proportionalto its activity. After revealing, the gel was washed then dried andanalysed by densitometry to quantify the different alkalineisophosphatases.

Example n° 2

Gel with the following composition:

Agarose 1% Tris 0.38 M Boric acid 0.06 M sodium azide 1 g/l Triton X 10010 g/l Nonidet NP 40 5 g/l

40 ml of demineralised water and 0.5 g of agarose were introduced into a100 ml Erlenmeyer flask. After boiling for 5 minutes with constantstirring, the agarose had dissolved to produce a perfectly clearsolution. The temperature of this solution was reduced to 50° C. in athermostatted bath. 10 ml of a concentrated buffer solution containing229.9 g/l of Tris, 18.55 g/l of boric acid, 5 g9l of sodium azide, 50g/l of Triton X 100 and 25 g/l of Nonidet NP 40 were introduced into a50 ml Erlenmeyer flask. This solution was maintained at 50° C. in thethermostatted bath.

The pre-heated buffer was added to the agarose solution. It washomogenised and maintained at 50° C.

5 ml of the above solution, removed with a pipette, was then uniformlypoured onto a 10×8 cm hydrophilic plastic sheet.

After gelling and stabilisation, the gel could be used. Fresh sera to beanalysed were deposited in 2 adjacent deposits, 2.5 cm from the edge, onthe cathode side. A 2 mg/ml solution of Wheat Germ Agglutinin (WGA)lectin was applied simultaneously and over the same period 5 mm in frontof one of the 2 deposits of each sample, i.e., between the sample andthe anode.

The alkaline isophosphatases were separated by electrophoresis in anapparatus which could adjust the temperature to 20° C. Migration wascarried out at a constant power of 20 W for 20 minutes.

The alkaline phosphatase activities and densitometry were measured as inthe previous example.

What is claimed is:
 1. An electrophoretic process for separatingalkaline phosphatase isoenzymes from a biological sample comprising thesteps of: depositing a solution of lectin onto an electrophoresissupport in a first localised zone, and depositing a biological sampleincluding alkaline phosphatase isoenzymes onto said electrophoresissupport in a second localised zone which is spaced apart from said firstlocalized zone and applying an electric field to said electrophoresissupport to permit electrophoretic separation by migration of saidisozymes from said second localized zone such that at least one of saidisozymes migrates through said first localized zone.
 2. The process forseparating alkaline phosphatase isoenzymes of claim 1, furthercomprising the step of quantitative analysis of at least one of saidisoenzymes following said separation.
 3. The process for separatingalkaline phosphatase isoenzymes of claim 1, wherein said lectin solutionand said biological sample are each deposited over a period of betweenabout 5 to 20 minutes.
 4. The process for separating alkalinephosphatase isoenzymes of claim 1, wherein the concentration of saidlectin solution deposited is determined based upon the migrationtemperature and the period over which said lectin solution is applied tosaid support.
 5. The process for separating alkaline phosphataseisoenzymes of claim 4, wherein said concentration of said lectinsolution ranges from between about 0.1 mg/ml to the limit of solubilitylectin in water.
 6. The process for separating alkaline phosphataseisoenzymes of claim 5, wherein said concentration of said lectinsolution ranges from between about 1 to 10 mg/ml.
 7. The process forseparating alkaline phosphatase isoenzymes of claim 4, wherein saidmigration temperature ranges from between 18° C. to 53° C.
 8. Theprocess for separating alkaline phosphatase isoenzymes claim 4, whereinsaid concentration of said lectin solution is determined as a functionof the maximum migration temperature achieved in the electrophoresissupport during said migration.
 9. The process for separating alkalinephosphatase isoenzymes of claim 1, wherein said electric field isapplied between an anode and a cathode and wherein said isozymes migratetoward said anode under the influence of said electric filed and whereinsaid first localized zone is located between said anode and said secondlocalised zone.
 10. The process for separating alkaline phosphataseisoenzymes of claim 1, wherein said lectin is deposited between thebiological sample and a position normally occupied by an intestinalfraction 3 (I3) at the end of said migration step.
 11. The process forseparating alkaline phosphatase isoenzymes of claim 1, wherein thelectin is wheat germ lectin.
 12. The process for separating alkalinephosphatase isoenzymes of claim 1, wherein said electrophoresis supportis an agarose gel or polyacrylamide.
 13. The process for separatingalkaline phosphatase isoenzymes of claim 1, wherein said electrophoresissupport is a cellulose acetate membrane.
 14. The process for separatingalkaline phosphatase isoenzymes of claim 2, wherein said quantitativeanalysis is carried out by measuring the densitometry of saidelectrophoresis support after staining.
 15. The process for separatingalkaline phosphatase isoenzymes of claim 1 wherein said alkalinephosphatase isoenzymes includes a osseous fraction, at least a potion ofwhich does not migrate through said first localised zone.
 16. Theprocess for separating alkaline phosphatase isoenzymes of claim 15wherein said alkaline phosphatase isoenzymes includes at least onehepatic fraction, which does migrate through said first localised zonethereby producing separation between said osseous and hepatic fractions.17. The process for separating alkaline phosphatase isoenzymes of claim2 wherein said alkaline phosphatase isoenzymes includes a osseousfraction, at least a potion of which does not migrate through said firstlocalised zone.
 18. The process for separating alkaline phosphataseisoenzymes of claim 17 wherein said alkaline phosphatase isoenzymesincludes at least one hepatic fraction, which does migrate through saidfirst localised zone thereby producing separation between said osseousand hepatic fractions.
 19. The process for separating alkalinephosphatase isoenzymes of claim 2 wherein the separated ALP isoenzymesare quantitatively analyzed.
 20. The process for separating alkalinephosphatase isoenzymes of claim 19, wherein said quantitative analysisis carried out by measuring the densitometry of said electrophoresissupport after staining.
 21. The process for separating alkalinephosphatase isoenzymes of claim 1, wherein said concentration of saidlectin solution ranges from between about 0.1 mg/ml to the limit ofsolubility lectin in water.
 22. The process for separating alkalinephosphatase isoenzymes of claim 2, wherein said lectin is depositedbetween the biological sample and a position normally occupied by anintestinal fraction 3 (I3) at the end of said migration step.
 23. Anelectrophoretic process for detecting the presence of at least onealkaline phosphatase isoenzyme in a biological sample deposited on anelectrophoretic support, comprising the steps of: depositing a solutionof lectin onto said electrophoresis support in a first localised zone,depositing a biological sample including at least one alkalinephosphatase isoenzyme onto said electrophoresis support in a secondlocalised zone which is spaced apart from said first localised zone suchthat at least one alkaline phosphatase isoenzyme contained within saidbiological sample will interact with said lectin as it migrates towardan electrode upon the application of an electric field to saidelectrophoresis support.
 24. The process of claim 23 further comprisingthe step of correlating the presence of said at least one alkalinephosphate isoenzyme with a hepatic or bilary disorder.
 25. The processof claim 23 further comprising the step of correlating the presence ofsaid at least one alkaline phosphate isoenzyme with an osseous disorder.